The present invention relates to a method of high sensitivity immunoassay, specifically to a method of ultrahigh sensitivity immunoassay for antigen-specific antibodies or antigenic substances.
Assay for antibodies is widely applied to examinations for infectious diseases, autoimmune diseases, etc., and it is important in examinations for autoimmune diseases, etc., to assay the component which has already formed an antigen-antibody complex in a test solution.
In addition, assay for biocomponents, in particular, antigenic substances in the body fluid of humans, is very important in clinical situations. Antigen assay is widely applied to various examinations, such as endocrine examinations by measurement of hormones, cancer diagnostic examinations and infection diagnostic examinations.
Such microdetermination for antibodies or antigenic substances has conventionally been based on immunoassay. In recent years, methods using a solid carrier have become widely used in immunoassay. Examples of such methods include methods based on sandwich technique, such as ELISA and IRMA, and methods based on competitive protein binding analysis, such as the second antibody solid phase method.
The conventional methods of immunoassay are divided into two groups: one comprises methods in which each specific antibody or antigen in the sample is trapped on an antigen- or antibody bound to a solid carrier and assayed using a labeled anti-immunoglobulin antibody or labeled antibody (art 1); the other group comprises methods in which each specific antibody is trapped on an anti-immunoglobulin antibody-coated carrier and assayed using a labeled antigen (art 2).
As an example of application of art 1, there is a report on a study of assay for human anti-insulin antibody by L. J. Nell et al., [Diabetes, 34, 60, (1985)], in which test solution was added to an insulin-bound solid carrier and bound human anti-insulin antibody was determined using enzyme labeled anti-human immunoglobulin antibody. As an example of art 2, there is a report on a study of assay for anti-toxoplasma IgM antibody by A. M. Johnson et al., [Pathology, 17, 586 (1985)], in which an anti-IgM antibody-coated solid phase was used.
In art 1, a large amount of nonspecific immunoglobulin is normally contained in the test solution, and it is absorbed nonspecifically to the solid phase to cause binding of the labeled immunoglobulin antibody; therefore, art 1 has disadvantage in that assay sensitivity decreases as a result of an increase in background value.
Also in the case of assay for antigenic substances, assay sensitivity is restricted by the background value attributable to a labeled component which is nonspecifically adsorbed onto solid carrier, and this poses a limitation on high sensitivity assay.
In art 2, there is a limitation on the capability of trapping immunoglobulin of anti-immunoglobulin antibody-coated solid carrier. Increase in carrier capability results in increase in background value. In any case, it is difficulty to obtain high sensitivity.